The most widely used protocol for the induction of experimental allergic airway inflammation in mice involves sensitization by intraperitoneal (i.p.) injections of the antigen ovalbumin (OVA) used in conjunction with the adjuvant aluminium hydroxide . Although adjuvants are frequently used, there are questions regarding the necessity of alum for murine asthma studies due to the non-physiological nature of this chemical.The objective of this study was to compare experimental asthma phenotypes between adjuvant and adjuvant-free protocols of murine allergic airway inflammation in an attempt to develop a standardized alternative to adjuvant use.An adjuvant-free OVA model of experimental asthma was investigated in BALB/c mice using i.p. or subcutaneous (s.c.) sensitization routes. For the s.c. sensitization, β-galactosidase  was also tested as an antigen. In addition, OVA adjuvant and adjuvant-free sensitization protocols were compared in BALB/c and C57BL/6 mice. Open-field testing was performed to assess the effect of alum on mouse behaviour.Comparison of adjuvant vs. adjuvant-free and i.p. vs. s.c. protocols revealed that both adjuvant use and route of antigen application significantly influenced OVA-specific antibody production. Comparison of adjuvant and adjuvant-free protocols in this study clearly demonstrated the non-requirement of alum for the induction of acute allergic airway inflammation, as both protocols induce a similar disease phenotype. BALB/c mice were significantly more susceptible than C57BL/6 mice to sensitization. Using the improved s.c. adjuvant-free protocol, it was demonstrated that alternative antigens such as β-gal can also be utilized. Behavioural studies indicated severe distress in mice treated with alum.The OVA s.c. adjuvant-free protocol used in this study generates a phenotype comparable to the benchmark adjuvant protocol widely used in the literature. The adjuvant-free alternative avoids the added complication of non-physiological adjuvants that may interfere with asthma treatment or prevention strategies. With regard to allergic bronchial asthma, mouse models provide an excellent means to examine phenotypes such as lung inflammation, airway reactivity, bronchoalveolar lavage (BAL) cell counts, antigen-specific antibody titres and the expression of inflammatory cytokines.Laboratory mice are widely utilized to study a multitude of diseases and are essential for the analysis of Several methods are used to induce experimental asthma in the mouse and the most frequently used protocol in the literature involves sensitization by intraperitoneal (i.p.) injections of antigen used in conjunction with the adjuvant aluminium hydroxide  . AlthougIn addition to the non-physiological nature of alum, this adjuvant is not required for the generation of an acute allergic inflammatory response using the antigen ovalbumin (OVA) . The litWith the recent resurgence of interest in murine experimental asthma models and the action of adjuvants, it is timely to include adjuvant-free alternatives in the discussion. The objective of this study is to optimize adjuvant-free sensitization with regard to a fully developed phenotype of allergic airway inflammation and clinical features of experimental asthma. To illustrate the effectiveness of the adjuvant-free protocol, comparison of the phenotypes generated by adjuvant and adjuvant-free protocols will be performed in BALB/c and C57BL/6 mice.ad libitum. All experimental procedures were approved by the local animal ethics committee and met German and international guidelines. At least eight animals were used per group.Female BALB/c and C57BL/6 mice aged 6–8 weeks were obtained from Harlan Winkelmann  and housed four animals per cage in a 12/12 h light/dark cycle with food and water available Investigations concerning the effect of adjuvant and route of sensitization were all performed using Protocol 1 see . The i.pAlternatively, mice were sensitized s.c. with 10 μg β-galactosidase  (Sigma) in 200 μL PBS or sham injections of PBS only and i.n. challenged on days 26, 27 and 28 with 50 μg β-gal in 50 μL PBS or sham PBS only  of bronchial responsiveness to β-methacholine was measured as described previously .Forty-eight hours after the last aerosol challenge, mice were terminally anaesthetized with ketamine plus rompun and blood samples were taken from the axillary vessels. The levels of OVA-specific IgE and IgG1 were measured by an enzyme-linked immunosorbent assay (ELISA)  in the serum as described previously .BAL was performed 48 h after the last challenge using 1 mL PBS containing 1 × protease inhibitor cocktail  as described in Neuhaus-Steinmetz et al. . An autoCell-free supernatants from BAL fluids were analysed for IL-5, IL-10, IL-13, TNF-α and IFN-γ content by sandwich ELISA as described previously in Herz et al. . The detDirectly after BAL, lungs were fixed with 10% formalin via the trachea, removed and stored in 10% formalin. Lung tissues were embedded into paraffin and 3 μm sections were stained with haematoxylin and eosin (HE) or periodic acid Schiff (PAS).http://www.biobserve.com).To determine the effect of alum on mouse behaviour, BALB/c mice were tested using an open-field test . AnimalsT-test (in the case of a two group comparison), one-way anova with Tukey's Multiple Comparison Test (for multiple group comparison) or two-way anova for comparison of multiple groups with two influencing factors (protocol vs. strain).Graphing and statistical analysis of normally distributed data was performed using Prism 5 . Data are expressed as mean ± SEM and are analysed for significance using Student's Using Protocols 1A and 1B , an initLooking further into the OVA adjuvant-free model, we also tested whether varying the site of sensitization induced changes in allergic sensitization; the differences between i.p. and s.c. routes of delivery were examined using Protocol 1B and 1C . CompariThe second part of the study focused specifically on comparison of the phenotype generated by OVA adjuvant and adjuvant-free protocols. Since it was previously determined that the s.c. adjuvant-free model is most effective with Protocol 1 and the i.p. adjuvant model is most effective with Protocol 2  (data noBoth the s.c. adjuvant-free (Protocol 1C) and the i.p. adjuvant (Protocol 2A) protocols induced an equal amount of inflammatory cell infiltration into the BAL fluid in both mouse strains. The influx of eosinophils and lymphocytes, which were predominantly responsible for the increase in leukocyte cell numbers, also showed no differences upon comparison of the two protocols, shown in Observation of HE-stained lung sections from OVA-sensitized and -challenged animals demonstrated that both s.c. adjuvant-free (Protocol 1C) and i.p. adjuvant (Protocol 2A) protocols had similar lung histology and perivascular inflammation regardless of the strain (data not shown). Comparison of PAS-stained lungs between mouse strains revealed that regardless of the protocol used, OVA-sensitized and -challenged BALB/c mice had larger numbers of mucus-producing goblet cells than C57BL/6 animals. C57BL/6 mice exhibited less mucus production compared with BALB/c mice regardless of the protocol used .P<0.001).As shown in P<0.001) and OVA-specific IgE production (P<0.001). This is in agreement with the previous investigation of antibody levels in relation to adjuvant use and site of injection, seen in As expected, observation of OVA-specific antibody titres from OVA-sensitized and -challenged mice demonstrated that the s.c. adjuvant-free protocol (Protocol 1C) resulted in significantly higher OVA-specific IgE and significantly lower OVA-specific IgG1 levels than the i.p. adjuvant protocol (Protocol 2A). Analysis of protocol vs. strain indicated that the protocol had a strong effect on both OVA-specific IgG1 (P<0.001) or adjuvant-free Protocol 1C  was used. Also in sensitized mice using the s.c. adjuvant-free Protocol 1C, BALB/c mice expressed higher OVA-specific IgG1 titres than C57BL/6 mice . This trend was not observed when using the i.p. adjuvant Protocol 2A; both BALB/c and C57BL/6 mice expressed equivalent levels of OVA-specific IgG1 .Interestingly, assessment of strain differences revealed that in OVA-sensitized mice, the BALB/c strain consistently produced higher OVA-specific IgE antibody titres than the C57BL/6 strain regardless of whether the adjuvant Protocol 2A  and showed a very significant influence of mouse strain on BAL fluid IL-10 levels (P<0.001). IFN-γ and TNF-α were not measurable in the BAL fluid of any of the samples tested.Cytokine analysis  showed tTo demonstrate the versatility of the s.c. adjuvant-free protocol for use with antigens alternative to OVA, BALB/c mice were s.c. sensitized and i.n. challenged with β-gal using Protocol 1D . Figure In the standardized open-field testing conducted, it was demonstrated that mice treated i.p. with the adjuvant alum displayed significantly decreased average velocity (movement) and rearing (awareness of surroundings) 15 min after treatment. This distress resolved within 2 or 4 h, respectively, Research investigating the underlying mechanisms of human disease, as well as the initial testing of therapeutic agents, depends heavily on animal models. Illustrating this, murine models are commonly used in asthma research and many different methods are used to generate acute allergic airway inflammation. On the whole, protocol selection and choice of mouse strain are paramount to the study design and it is desirable to mimic the natural physiology as closely as possible. In contrast to this, the most frequently used method to sensitize mice to allergen utilizes the non-physiological adjuvant alum to promote the development of a TH2-driven immunological response , 28.Although alum is regularly used in mouse models of experimental asthma, its mechanism of action has only recently begun to be elucidated. Phagocytosis of alum plus antigen has been shown to result in phagosomal destabilization , 30, actRegardless of the mechanism by which alum acts as an adjuvant, there is a large body of evidence in the literature demonstrating that adjuvant is not necessary for the induction of murine experimental asthma , 32–35. With the use of an adjuvant-free protocol, it is speculated that additional danger signals, such as LPS content in the OVA preparation, may be required to facilitate the sensitization process . Indeed,Subsequent comparisons of the new OVA s.c. adjuvant-free protocol with an OVA i.p. adjuvant protocol frequently used in the literature demonstrated that with the exception of increased IL-5 production and differences in OVA-specific antibody titres, the s.c. adjuvant-free protocol produces an allergic airway inflammation phenotype comparable with the standardized i.p. adjuvant protocol. As it is well known that alum participates in the generation of humoral immunity , the difComparison of the inflammatory phenotype generated by the i.p. adjuvant and s.c. adjuvant-free protocols in different strains of mice supports the notion that the choice of the mouse strain is a crucial aspect for experimental design. With regard to OVA-induced airway inflammation, we demonstrated that the magnitude of immune response with respect to goblet cell hyperplasia, airway reactivity, BAL fluid IL-5, IL-10 and serum OVA-specific IgE production is significantly lower in C57BL/6 mice than in BALB/c. These findings were similar regardless of whether an i.p. adjuvant or an s.c. adjuvant-free protocol was used. These findings support many others in the literature that have found lower acute inflammatory responses in C57BL/6 mice , 38–40.During use of the i.p. adjuvant protocol, abnormal mouse behaviour was observed beginning 15 min after OVA alum injection. To investigate this further, we sought to quantify the effect of alum on mice using standardized, quantitative behavioural analysis. Open-field testing, which is used to assess anxiety, alertness, locomotor activity and exploration , revealeIn conclusion, this research has demonstrated that the improved OVA s.c. adjuvant-free protocol used in this study generates a phenotype similar to the standard OVA i.p. adjuvant protocol used in the majority of studies investigating mechanisms of respiratory allergy in mice. For researchers with concerns about the non-physiological nature of alum, we demonstrate that the s.c. adjuvant-free model is an excellent alternative to adjuvant use.


Understanding what constitutes an important difference on a HRQL measure is critical to its interpretation. The aim of this study was to provide a range of estimates of minimally important differences (MIDs) in EQ-5D scores in cancer and to determine if estimates are comparable in lung cancer.A retrospective analysis was conducted on cross-sectional data collected from 534 cancer patients, 50 of whom were lung cancer patients. A range of minimally important differences (MIDs) in EQ-5D index-based utility (UK and US) scores and VAS scores were estimated using both anchor-based and distribution-based (1/2 standard deviation and standard error of the measure) approaches. Groups were anchored using Eastern Cooperative Oncology Group performance status (PS) ratings and FACT-G total score-based quintiles.For UK-utility scores, MID estimates based on PS ranged from 0.10 to 0.12 both for all cancers and for lung cancer subgroup. Using FACT-G quintiles, MIDs were 0.09 to 0.10 for all cancers, and 0.07 to 0.08 for lung cancer. For US-utility scores, MIDs ranged from 0.07 to 0.09 grouped by PS for all cancers and for lung cancer; when based on FACT-G quintiles, MIDs were 0.06 to 0.07 in all cancers and 0.05 to 0.06 in lung cancer. MIDs for VAS scores were similar for lung and all cancers, ranging from 8 to 12 (PS) and 7 to 10 (FACT-G quintiles).Important differences in EQ-5D utility and VAS scores were similar for all cancers and lung cancer, with the lower end of the range of estimates closer to the MID, i.e. 0.08 for UK-index scores, 0.06 for US-index scores, and 0.07 for VAS scores. It is common, if not usual practice, to include health-related quality of life (HRQL) measures in clinical trials in oncology. To justify the cost of new cancer drugs, decision-makers need to determine not only whether a drug has a statistically significant impact on survival and/or HRQL, but they also need to evaluate whether the improvement is meaningful. This is particularly important in lung cancer, where aggressive new therapies are being brought to market. In addition to the use of cancer-specific measures such as European Organization for Research and Treatment of Cancer-QLQ-C30 (EORTC QLQ-C30) ,2and theA major challenge in HRQL measurement is the interpretation of scores, particularly with respect to defining what constitutes a minimally important difference (MID). The MID has been defined as the smallest change in a PRO measure that is perceived by patients as beneficial or that would result in a change in treatment . ApproacWhile MIDs have been estimated for EQ-5D index-based scores for some conditions , empiricA retrospective analysis was conducted on cross-sectional data collected from 534 cancer patients with eleven types of cancer who participated in a validation study of cancer symptoms scales . ParticiThe patients were recruited from six sites within the National Cancer Coalition Network (NCCN) and the Cancer Health Alliance of Metropolitan Chicago (CHAMC). The NCCN is a not for profit, tax-exempt corporation that is an alliance of National Cancer Institute (NCI) approved comprehensive cancer centers. The CHAMC organizations provide social, emotional and informational support services to cancer patients free of charge. These organizations are not affiliated with a medical center or university, and each CHAMC agency serves different geographical and socio-demographic cancer patient populations. All patients who completed the questionnaires consented to participate in the study. Institutional review board approval was obtained for secondary data analysis (University of Illinois at Chicago research protocol #2006-0891).Patients completed several questionnaires, including the EQ-5D and the Functional Assessment of Cancer Therapy (FACT). The EQ-5D descriptive system consists of 5 dimensions: Mobility, Self-Care, Usual Activities, Pain/Discomfort, and Anxiety/Depression, each with 3 levels  . Index-bParticipants also completed the Functional Assessment of Cancer Therapy (FACT) quality of life questionnaire using a version specific to their tumor type. The general subscales common to all versions (FACT-G) include physical well-being (PWB), social/family well-being (SFWB), emotional well-being (EWB), and functional well-being (FWB). The FACT-G total score  is based on 26 summed items (responses 0 to 4) from the PWB (7 items), FWB (7 items), SFWB (6 items), and EWB (6 items). Higher scores represent better quality of life.Performance status was evaluated using the Eastern Cancer Oncology Group (ECOG) classification system . ECOG grBoth anchor-based and distribution-based approaches were used to estimate MIDs for the EQ-5D in the overall cancer cohort, and in the subgroup of lung cancer patients, when possible. Distribution-based criteria included: 1/2 standard deviation (SD) and the standard error of the measure (SEM) . For conn cancer . Becausen cancer . CalculaAnchors can be constructed using clinically-based criteria, such as response to treatment, or more subjective criteria, e.g. health status. We used ECOG grades, assessed by physician, to group patients into categories of performance status, and determined mean difference scores between ECOG grades. Distribution-based criteria were then applied to the statistics associated with each anchor-based category. A second anchor-based approach used FACT-G scores. The cohort was stratified into quintiles based on FACT-G summary scores. Grouping the cohort into quintiles approximated an appropriate threshold for stratifying patients based on MID estimates for the FACT-G, have been identified as close to 6 in previous studies: 6–7 in hepatobiliary carcinoma , and 5–6Similar demographic characteristics were observed in the overall cancer sample and the lung cancer subgroup Table . A wide For all cancer patients, mean difference scores anchored by ECOG status ranged from 0.09 to 0.16 for UK scores and from 0.07 to 0.11 for US scores Table . Across UK = 0.10, 1/2 SDUK = 0.09; SEMUS = 0.07, 1/2 SDUS = 0.06. For the lung cancer subgroup, average MIDs between quintiles were 0.10 (UK) and 0.07 (US), with SEMUK = 0.09, 1/2 SDUK = 0.08; SEMUS = 0.06, 1/2 SDUS = 0.06.Average mean estimates of MIDs across FACT-G based quintiles for the overall cancer cohort were 0.09 for UK scores, 0.06 for US scores Table . Using dMID estimates for EQ-5D VAS scores based on FACT-G score quintiles were the same for both the overall cancer groups and the lung cancer subgroup (Table Interpretation of scores is an important issue in the field of HRQL measurement, but there is no consensus on the most appropriate method for assessing the ability of an instrument to capture meaningful differences. In this study, we followed criteria established in previous investigations of MIDs -15. We fThe issue of what constitutes an MID on a measure of HRQL is part of an ongoing dialogue about issues of interpretation. Developers of HRQL measures have not been forthcoming in the literature in explicitly attempting to establish MIDs. One reason to avoid this is because clinically important differences may vary with the target population. Limitations in the scaling properties of a measure can contribute to inconsistent MID estimates, as they may depend upon where a patient or group falls along the continuum of the measure. Distribution-based approaches for estimating important differences rely on the assumption of normality, and ceiling effects particularly in healthier patient populations produce skewed score distributions. Although ceiling effects have been associated with the use of EQ-5D , a ceiliMID estimates for EQ-5D in this study can be compared to other studies that have examined important differences using EQ-5D. A previous study by Walters and Brazier compared minimally important differences between SF-6D and EQ-5D, and reported a mean MID of 7.4 for the UK-based algorithm . Their eMIDs are often estimated using longitudinal datasets, and difference scores based on changes over time were not available in this dataset, which was cross-sectional. However, the MIDs for EQ-5D UK-based utility scores reported using longitudinal data  were comAn additional issue for users of EQ-5D is the selection of preference-based algorithm. As observed in this study, MIDs varied with the selection of the algorithm. MIDs for EQ-5D UK index-based utility scores ranged from 0.08 to 0.16 with a mode of 0.10. For US-based scores, a range of 0.06 to 0.12 was reported, with a mode of 0.07. This result was not unexpected, as the US preference-based algorithm produces scores with a smaller range than the UK scores, resulting in smaller difference scores and smaller standard deviations, thus smaller MIDs.In summary, important differences in EQ-5D summary scores were similar for all cancers and lung cancer, with the lower bounds likely to represent a closer estimate of true MID, i.e. 0.08 for UK-based scores, 0.06 for US-based scores, and 0.07 for VAS scores. MIDs for EQ-5D UK-based utility scores in cancer were similar to estimated MIDs for other conditions in the published literature. To our knowledge, MIDs for EQ-5D VAS scores and US-based utility scores have not been previously reported. Across the different approaches, MIDs for US-based utility scores were consistently smaller than MIDs for UK-based utility scores.MID – minimally important differenceHRQL – health-related quality of lifeFACT-G – Functional Assessment of Cancer Therapy – GeneralSEM – standard error of the measureSD – standard deviationPWB – physical well-being,SFWB – social/family wellbeingEWB – emotional well-beingFWB – functional well-beingECOG – Eastern Cancer Oncology GroupPS – performance statusA. Simon Pickard is a member of the executive committee of the EuroQol group, a not for profit group that developed and distributes the EQ-5D. David Cella is developer of the FACIT measurement system. Drs. Pickard and Cella have received consulting fees from GlaxoSmithKline, which financed this manuscript including the article-processing charge. They do not have any stocks or shares in an organization that may gain or lose financially from the publication of this manuscript.ASP, MN and DC were responsible for the conception of the study. ASP and DC acquired the data. ASP performed the data analysis and drafted the manuscript. MN and DC revised it critically for intellectual content, and all authors approved of the final version.


Human immunodeficiency virus type 2 (HIV-2) emerged following cross-species transmission of simian immunodeficiency virus (SIV) from sooty mangabeys to humans several decades ago. The epidemic groups of HIV-2 have been established in the human population for at least 50 years. However, it is likely that new divergent SIVs can infect humans and lead to new outbreaks. We report the isolation of a new strain of HIV-2, HIV2-NWK08F, from an immunodeficient Sierra Leone immigrant. Health care providers in Sierra Leone and elsewhere need to be alerted that a subtype of HIV-2, which is not detected by PCR for epidemic HIV-2 strains, exists and can lead to immunosuppression. Infection with human immunodeficiency virus type 2 (HIV-2) is endemic in some countries of West Africa. Unlike infection with HIV type 1 (HIV-1), this infection has not appreciably spread beyond this area. The incidence of HIV-2 infection has even declined over the last 16–20 years . HoweverSixteen years ago, infection with HIV-2 Group F was described in one individual from the northern province of Sierra Leone. HIV-2 GPatient X is a 68 year old male from Freetown, Sierra Leone. Patient X immigrated to the New Jersey, USA. in 2007. During the immigration process, he tested positive for antibodies against HIV. He was referred to the Peter Ho Memorial Clinic in Newark, New Jersey for follow-up and treatment in early 2008. Patient X's serum was repeatedly reactive by serological testing with ELISA kits containing HIV-1 and HIV-2 antigens. The western blot for HIV-1 was negative. His HIV-1 viral load was <48 copies and polymerase chain reaction (PCR) for HIV-1 proviral DNA was negative. An HIV-2 immunoblot was positive. The presumptive diagnosis was that Patient X had an HIV-2 infection. However, a PCR assay from a commercial laboratory for HIV-2 proviral DNA was negative . This result suggested one of two possibilities:1. The proviral load was below the limit of detection of the assay.2. The virus was too divergent from known HIV-2 epidemic groups to be amplified by the gag primers based on epidemic subtype consensus sequence.env and gag of this provirus with subtype F primers. To rule out the possibility of PCR contamination, the env region was independently amplified in two laboratories, one in Newark, New Jersey and the other in Covington, Louisiana. The env sequence data were identical. Figure gag. HIV-2NWK08F clusters significantly with six other viruses, all from Sierra Leone. Two viruses were found in household pet sooty mangabeys which are native to the region. A third was HIV-2 subtype E; a fourth was subtype F, from a woman who lived in the northern province of Sierra Leone – the same area as the original home of patient X.Patient X had a CD4 T-cell count of 338 cells/μl and a CD4:CD8 ratio of 0.52. This CD4 T-cell lymphopenia suggested that Patient X was actively infected with a divergent strain of HIV-2. To determine if Patient X had active infection with a non-epidemic strain, we attempted to isolate the virus and performed PCR with primers that were used in our previous study of HIV-2 in Sierra Leone. On fourenv primers and probe. Patient X had a proviral load equal to 6,100 copies per 106 PBMC.Subtype F HIV-2 has not previously been known to cause immune suppression nor has it been known to be transmitted from person to person. It is not known how patient X acquired HIV-2-NWK-08F. Patient X denied exposure to monkeys. He denied ever hunting game. He had no tattoos, no history of needle exposure in Sierra Leone and no history of blood or blood product transfusion. Patient X reported only one sexual contact, his wife. No relative was available for testing. HIV-2-NWK-08F clusters most closely with HIV-2SL93F and next most closely with the 2 SIVs found in sooty mangabeys in Sierra Leone Figure . A real 6 PBMC[It is alarming that Patient X's virus was easily isolated and that his CD4 T-cell count is decreased with an abnormal CD4:CD8 ratio. Patient X's reported lack of exposure to pet monkeys or by hunting is also a concern, since it implies human to human transmission. Two recent studies of HIV-2 infected individuals found the median proviral load to be ~300 copies per 106 PBMC. The proFurthermore, the commercial assay for establishing the existence of active infection, namely PCR for HIV-2 proviral DNA, did not detect the provirus of this isolate. This result, similar to problems with early viral load assays measuring non-subtype B HIV-1 viremia, indicatThese data demonstrate that a pathogenic, novel strain of HIV-2 is circulating, at least, within Sierra Leone. Health care providers in Sierra Leone and elsewhere need to be alerted that a strain of HIV-2, which is not detected by PCR for epidemic HIV-2 strains, exists and can lead to immunosuppression. Epidemiologic studies are required to determine the extent of this virus' spread in Sierra Leone and to other countries.Verbal consent was obtained from this patient by SMS. The consent was witnessed by VDL. The consent is available for review by the Editor-in-Chief of Retrovirology.The authors declare that they have no competing interests.env. LA recognized the possibility that the patient was HIV-2 infected and provided valuable demographic data. RG, AG, CA, and PAM amplified gag and performed the phylogenic analysis. All authors read and approved the final manuscript.SMS conceived of the study, designed most of the experiments and wrote the manuscript. DE isolated the virus. VDL developed the real-time PCR protocol. US assisted with cloning


Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots. Of all known protein post-translational modifications (PTM), glycosylation is indeed the most common and complex one, which is predicted to occur on over 50% of all proteins and shows a great of diversitiesvia an N-acetylglucosamine residue, and Xxx can be any amino acid except proline. After digestion with PNGase F, the NH2 group in asparagine changes into OH group at the glycosylation site, and the mass of deglycosylated peptides increases 0.984 Dalton, which can be detected unambiguously with MALDI-TOF. In mammals, sialic acids are added by the capping reaction, and they are very important in cell-cell interactions. It is reported that the number of sialic acid moieties changes significantly during acute and chronic diseases, indicating that sialylated glycoproteins may be biomarker candidates I values in 2-DE due to the different number of terminal sialic acids Presently, there are four types of known glycosylation, in which N-glycosylation and O-glycosylation are the most common ones. N-glycosylation occurs on asparagine residues in the consensus sequence Asn-Xxx-Ser/Thr  Generally, glycoprotein characterization includes several steps, i.e. protein identification, determination of glycosylation sites and evaluation of site-specific microheterogeneities. Larsen et al. introduced a fast and sensitive method to characterize gel-separated nonsialylated N-glycoproteins Here we introduced an improved protocol to enhance the analysis of large glycopeptides over 4000 Dalton and low-abundance glycopeptides of which the site-specific glycosylation could not be determined with MS/MS. In this study, the composition of these glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. The sequential deglycosylation and desialylation greatly improve the detection of deglycosylated peptides and desialylated glycans. In addition, improved peptide desalting and neutral glycan identification methods were utilized in the protocol, which further enhanced its overall sensitivity. It was verified with 10 pmol gel-separated human transferrin and α1-antitrypsin spots from 2-DE separated human serum proteins.The use of human sera for proteomics research was approved by the Institutional Review Board (IRB) of Beijing Institute of Genomics and informed consent was given by the participant in written.Human transferrin, α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (DHB), 2,4,6-trihydroxyacetophenone Monohydrate (THAP), diammonium citrate (DAC) and PNGase F were purchased from Sigma. Sialidase was purchased from Calbiochem. To pack microcolumns, HILIC material , Poros R2 material  and graphitized carbon ) were packed in constricted GELoader tips  as described For one-dimensional electrophoresis, 5 pmol and 10 pmol human transferrin was loaded, respectively. For 2-DE, 10 µL untreated human serum was loaded to each strip  for analyzing human α1-antitrypsin. The isoelectric focusing and 2-DE PAGE were conducted as described Peptides and glycopeptides were extracted from gel particles under sonication for 20 minutes with 50% ACN containing 0.1% TFA twice 4HCO3, pH 8.0. To the solution, 1 µL PNGase F (5 U/100 µL) was added and incubated at 37°C overnight. One microliter of the digested samples was used to identify deglycosylated peptides on Bruker Anchorchip™ target.The rest of the HILIC enriched glycopeptides from the above step were dried in vacuo and redissolved in 20 µL 20 mM NH2, pH 5.5, 5 µL sialidase (1 U/mL) was added. It was then incubated at 37°C overnight for complete removal of sialic acids.After the above-mentioned PNGase F digested samples passed through a Poros R2 microcolumn, the flow-through was treated with 0.05N HCl at 70°C for one hour to remove sialic acids The glycan purification procedure followed previously described method The CHCA matrix was prepared by dissolving 4 mg of CHCA in 1 mL of 70% ACN/0.1% TFA. The DHB matrix was prepared by dissolving 10 mg of DHB in 1 mL of 50% ACN/0.1% TFA. The THAP matrix was prepared by dissolving 7.5 mg of THAP in 1 mL of ACN/20 mM ammonium citrate (1∶1 v/v).−3 Torr) after mixed adequately with pipette tips. After vacuum drying, the sample appeared translucent. Upon absorption of moisture, the sample formed small crystals For peptides, 0.5 µL samples were applied on AnchorChip™ target . After they were dried, about 0.1 µL CHCA matrix was added. The dried target was desalted by washing with 0.1% TFA twice and subjected to MS analysis. Glycopeptide samples were prepared as above mentioned. For glycans, 0.5 µL samples were applied to Bruker #25184 MALDI target, and then 0.5 µL of THAP was added. They were dried under vacuum (50×10MALDI-MS (/MS) data were obtained by an Ultraflex mass spectrometer . For peptide mass fingerprint (PMF) identification, the mass spectrometer was externally calibrated in order to reach the mass accuracy of 100 ppm. The database searches were performed against NCBInr database using Mascot software with fixed modification as carbamidomethyl (C) and variable modifications as oxidation (M). Desialylated glycans were analyzed in the same way with peptides. Glycopeptides were analyzed in the m/z range of 2000–7000 in the linear positive-ion mode. External calibration was performed using the average values of 5 tryptic peptides or glycopeptides of horseradish peroxidase, and the mass accuracy of <0.05% was obtained. Typically, spectra from 200 laser shots were added to obtain the final specta. The mass signals were manually annotated. MS/MS analysis in the TOF/TOF mode was conducted as described http://www.expasy.org/tools/glycomod/). The masses of peptides and desialylated glycans were obtained in monoisotopic value in the reflectron mode of MALDI-TOF. However, because the masses of glycopeptides were obtained in average value in the linear mode of MALDI-TOF, the monoisotopic value of peptides and desialylated glycans must be turned into average values before calculation. Thus, we annotated their spectra manually with specific annotation method in order to get that average values. Finally, if the measured mass of a glycopeptide was equal to or 291*X  more than the predicted mass of a potential glycopeptide, i.e. different combinations of deglycosylated peptides and desialylated glycans, the potential glycopeptide should really exist, and the number of terminal sialic acids was X. The calculation is based on the fact that a glycopeptide is actually composed of three parts, i.e. the peptide part, the glycan part with sialic acids removed and the released sialic acids. The acceptable error was 500 ppm in the calculation.Estimation of N-linked glycan composition was accomplished by input of the peak masses into the GlycoMod Tool , we predicted potential glycosylation sites and the theoretical mass of the corresponding deglycosylated peptides from the sequence, i.e. 1477.744 (Asn-432), 3530.654 , 700.328 (Asn-491) and 2516.117 (Asn-630). In the following, the prediction would be confirmed by MS or MS/MS analysis on deglycosylated peptides.When glycopeptides enriched from gel-separated human transferrin were analyzed with MALDI-TOF, the mass spectra shown in pep+H−17]+, [Mpep+H]+, [Mpep+H+83]+ and [Mpep+H+203]+pep+H]+ can be determined, which leads to the identification of glycosylation sites. As shown in As shown in By comparing the masses of deglycosylated peptides in We got the mass spectrum in PG , MPG+291, MPG+291*2 and MPG+291*3 respectively. After comparing these values with the measured masses of glycopeptides from human transferrin, most glycopeptides were identified except for m/z 5234.1 and 5525.9. Base on the difference between m/z 5234.1 and 5088.7 and that between m/z 5234.1 and 5525.9, they were also identified (See also The calculation method mentioned in “See also . The numI to high pI. N-glycans on all three glycosylation sites of human α1-antitrypsin were determined successfully for both spots using our improved protocol. The mass spectra of the glycopeptides of A1PI-1 and A1PI-2 were listed between 5500 and 7000 Dalton in I for the same protein, which was consistent with previous research work In order to prove that our protocol was also applicable to 2-DE separated samples, we applied it to human α1-antitrypsin from 10 µL normal human serum separated by 2-DE . The twoBoth N-glycosylation sites and site-specific microheterogeneities were determined unambiguously from 10 pmol gel-separated human transferrin band using the improved protocol. We also tried 5 pmol samples as the lower limit. Although glycopeptides could also be identified, it was insufficient for the subsequent deglycosylation and desialylation. It was a little difficult to get 10 pmol samples from 2-DE gel spots, but sufficient samples could be obtained for glycosylation analysis by applying different amount of samples on gels for protein identification and glycosylation analysis, respectively In the past, many researchers used to desalt peptides by Poros R2 or graphite powder before MALDI-TOF analysis. It is a simple and convenient method. However, in this study, Bruker Anchorchip™ target was utilized in desalting peptides because its workflow was more simple and time-saving, which is consistent with the great number of proteomic protein identification. Furthermore, it is better in preserving small hydrophilic peptides that are generally lost during the desalting with other methods. Thus, higher protein sequence coverage was obtained in PMF identification Papac et al. reported that 50 fmol sialylated glycans were detected with signal-to-noise ratio of better than 5∶1 with MALDI-TOF when THAP was used as the matrixGenerally, many researchers used DHB or Super-DHB as the matrix in the analysis of neutral glycans One disadvantage of our protocol is that it provides no direct information about monosaccharide type and glycan linkage type. Generally, it needs to conduct permethylation, NMR research or sequential glycosidase digestion on glycans in order to get the detailed information, but gel-separated samples are surely insufficient for the treatments. In our protocol, glycan composition was determined accurately. Considering the biosynthesis process of glycans and known glycan structures in public databases, we could also get their structures. The site-specific glycosylation of large glycopeptides and low-abundance glycopeptides was mainly deduced from MS data but not from MS/MS due to the limitation of current mass spectrometers. In future, it is expected that MS/MS can be successfully done on these glycopeptides with MALDI-TOF/TOF with the quick development of mass spectrometers.Our protocol has extended the previous protocol to the analysis of large glycopeptides over 4000 Dalton and low-abundance glycopeptides. The sequential deglycosylation and desialylation improve the identification of glycosylation sites and glycans greatly. It is sensitive enough to analyze gel-separated sialylated glycoproteins, especially different glycoforms of the same glycoprotein separated by 2-DE. It can also be used in the glycosylation analysis of purified or recombinant glycoproteins due to its excellent sensitivity. It is reported that many potential biomarkers for cancers and inflammation are glycoproteins with different glycosylation status


Yeast two-hybrid (Y2H) screens have been among the most powerful methods to detect and analyze protein-protein interactions. However, they suffer from a significant degree of false negatives, i.e. true interactions that are not detected, and to a certain degree from false positives, i.e. interactions that appear to take place only in the context of the Y2H assay. While the fraction of false positives remains difficult to estimate, the fraction of false negatives in typical Y2H screens is on the order of 70-90%. Here we present novel Y2H vectors that significantly decrease the number of false negatives and help to mitigate the false positive problem.We have constructed two new vectors (pGBKCg and pGADCg) that allow us to make both C-terminal fusion proteins of DNA-binding and activation domains. Both vectors can be combined with existing vectors for N-terminal fusions and thus allow four different bait-prey combinations: NN, CC, NC, and CN. We have tested all ~4,900 pairwise combinations of the 70 Varicella-Zoster-Virus (VZV) proteins for interactions, using all possible combinations. About ~20,000 individual Y2H tests resulted in 182 NN, 89 NC, 149 CN, and 144 CC interactions. Overlap between screens ranged from 17% (NC-CN) to 43% (CN-CC). Performing four screens (i.e. permutations) instead of one resulted in about twice as many interactions and thus much fewer false negatives. In addition, interactions that are found in multiple combinations confirm each other and thus provide a quality score. This study is the first systematic analysis of such N- and C-terminal Y2H vectors.Permutations of C- and N-terminal Y2H vectors dramatically increase the coverage of interactome studies and thus significantly reduce the number of false negatives. We suggest that future interaction screens should use such vector combinations on a routine basis, not the least because they provide a built-in quality score for Y2H interactions that can provide a measure of reproducibility without additional assays. The yeast two-hybrid (Y2H) system has been among the most powerful methods to identify protein-protein interactions. However, it has also been criticized for generating large numbers of false positive and false negative data. While false positives can be minimized by including controls, retesting by independent methods, and bioinformatic filtering, false negatives pose a much bigger problem. In fact, many interactions must go undetected in two-hybrid screens because of the sterical constraints the system involves: the two fusion proteins must interact with each other, their interaction interfaces must be exposed, and all components must be oriented on the DNA in a way that the activation domain of the prey can productively interact with the transcriptional machinery.On top of sterical constraints, small variations of the Y2H system can lead to large differences in resulting interactions . As no oHere we present new vectors that vary one parameter of Y2H screening, namely the location of the Gal4 DNA-binding (DBD) and activation domain (AD) Figures  and 2. TEach of the four bait/prey vector combinations produced significantly different interactions. For example, the screens with bait ORF19, the large subunit of ribonucleotide reductase, produced a total of 17 interactions (of 15 distinct proteins), of which only two were found in all four combinations (namely ORF25 and ORF18C) Figure . Five inThe interactions detected also depended strongly on the selection pressure: typically all screens were initially carried out without 3-aminotriazole 3AT), a competitive inhibitor of imidazoleglycerolphosphate (IGP) dehydratase, i.e. the His3 reporter enzyme used in our assays. If baits turned out to be autoactivating under these conditions, we raised the 3AT concentrations up to 50 mM in steps of 1, 3, 10, 25, and 50 mM. ORF19 was an activator at 0, 1, and 3 mM and clear results were only obtained at 10 or 25 mM Figure . In geneT, a compAltogether, we have conducted more than 20,000 individual Y2H tests . Again, given that not all interactions are conserved, we do not expect to find all of these interactions. If single screens are performed (i.e. with only one of the 4 vector pairs) we find 14% of these core interactions on average  in all cases ranges from 45% to 50%  proteins, even if the NC combination yielded a significantly lower number of interations than the CN combination.Two-hybrid interactions have often been considered as unreliable, generating many false positive and false negative results . Often fObviously many more variations of the Y2H system are possible in addition to changing the location and nature of fusion tags, e.g. copy number, yeast strains, reporters etc. . Other YThe parental vectors and the two new C-terminal vectors described here are identical in most ways, including their origins of replication (and thus their copy number), their promoters (and thus protein expression levels), as well as the yeast strains in which they were expressed. In addition, the experimental conditions of our Y2H assays were identical (except for varying 3AT concentrations). Hence, we conclude that differences in results must result from structural constraints imposed by the location of the DBD and AD fusion tags and the associated sequences.Interactions that are found in multiple permutations but have not been reported in the literature are the most interesting ones for follow-up studies. For space reasons we cannot discuss all interactions that we consider as well substantiated by multiple Y2H assays. One intriguing example is the interaction between ORF57 and ORF10 which we found in all four permutations. ORF10 protein is the homologue of the herpes simplex virus type 1 (HSV-1) VP16 protein. Both proteins have been shown to be structural components of the virion tegument, but also appear to activate viral immediate-early (IE) protein promoters . In contLittle is known about the function of the VZV ORF57 protein. This protein has been shown to localize to the cytoplasm of infected cells and to be dispensable for the replication of VZV in vitro . HoweverTable S1). They should be excellent candidates for further studies of VZV molecular and structural biology.Our data contains a total of 15 pairs that are found in three or four permutations but have never reported in the literature . As with conventional screens bait constructs would have to be tested for auto-activation but this remains straight-forward. In order to improve mating efficiency the four combinations  could be mated separately and then mixed for further selection of positives. However, for less complex libraries (as those of virus genomes) this should not be necessary. Given that other factors in addition to N- or C-terminal fusions play a role in Y2H screens , additioIn summary, using permutations of fusion proteins has the potential to dramatically reduce false negatives in yeast two-hybrid screens while providing a simple way to rank interactions qualitatively. This should significantly improve future large-scale screens as well as downstream analysis of selected interactions.Literature-curated interactions were derived from : First, Here we describe a new set of vectors that are based on the well-established pGBKT7 and pGADT7 vectors (Clontech) and their Gateway derivatives pGBKT7g and pGADT7g  Figure . We modiWe systematically tested all N- and C-terminal baits against all N- and C-terminal VZV preys using standard matrix-based Y2H protocols as described previously ,22. The The authors declare that they have no competing interests.TS carried out all cloning steps and Y2H assays. TS, RH, AB, JH, and PU analyzed data. AB, VRP, and JH cloned the VZV ORFs. PU conceived this study. PU and AB wrote the manuscript. All authors read and approved the final manuscript.Table S1. Y2H data - all interactions described in this paper. A detailed legend is included in the Excel spreadsheet. The protein interactions from this publication have been submitted to the IMEx http://imex.sf.net consortium through IntAct (pmid: 17145710) and assigned the identifier IM-11718. The dataset is available at http://www.ebi.ac.uk/intact/search/do/search?searchString=IM-11718. Table S2. All PPIs - interactions of this study combined with all previously published interactions among herpesviral proteins. Table S3. Verifications. A. Permutations and contribution to interaction data. B. Verification by additional evidence. C. All permutations seem to be created equal. This table also contains more detailed explanations. Table S4. All VZV proteins and fragments used in this study.Click here for file


Women's Health Initiative randomized trials of postmenopausal hormone therapy reported intervention effects on several clinical outcomes, with some important differences between estrogen alone and estrogen plus progestin. The biologic mechanisms underlying these effects, and these differences, have yet to be fully elucidated.Baseline serum samples were compared with samples drawn 1 year later for 50 women assigned to active hormone therapy in both the estrogen-plus-progestin and estrogen-alone randomized trials, by applying an in-depth proteomic discovery platform to serum pools from 10 women per pool.P < 0.05) of change in concentration between baseline and 1 year for one or both of estrogen-plus-progestin and estrogen-alone groups. Quantitative changes were highly correlated between the two hormone-therapy preparations. A total of 98 proteins had false discovery rates < 0.05 for change with estrogen plus progestin, compared with 94 for estrogen alone. Of these, 84 had false discovery rates <0.05 for both preparations. The observed changes included multiple proteins relevant to coagulation, inflammation, immune response, metabolism, cell adhesion, growth factors, and osteogenesis. Evidence of differential changes also was noted between the hormone preparations, with the strongest evidence in growth factor and inflammation pathways.In total, 378 proteins were quantified in two or more of the 10 pooled serum comparisons, by using strict identification criteria. Of these, 169 (44.7%) showed evidence (nominal Serum proteomic analyses yielded a large number of proteins similarly affected by estrogen plus progestin and by estrogen alone and identified some proteins and pathways that appear to be differentially affected between the two hormone preparations; this may explain their distinct clinical effects. P < 0.05) compared with those for E-alone for other outcomes, including coronary heart disease (CHD) [versus risks [Postmenopausal hormone therapy was shown to have multiple effects of public-health importance in the Women's Health Initiative (WHI) randomized, placebo-controlled hormone-therapy trials of 0.625 mg/day conjugated equine estrogen   or of thse (CHD) ,8, breasse (CHD) ,10, and se (CHD) ,12, and us risks .Several of the articles just cited formally examined whether interactions occurred between the hormone-therapy hazard ratios and baseline study-subject characteristics. Although some moderate variations were detected , these It follows that much remains to be explained about the pattern of biologic changes induced by these hormone-therapy preparations in relation to the outcome effects mentioned earlier. Proteomic discovery work has the potential to identify biomarkers that may help to explain E+P or E-alone clinical effects or differences in effects between the two preparations. Hence, we applied a comprehensive quantitative proteomic approach designated Intact Protein Analysis System (IPAS) -19 to coWe recently reported  proteomiHere, we sought to uncover proteins and pathways that are differentially affected by E+P therapy relative to E-alone that would provide leads for the comparatively unfavorable effects with E+P observed in these trials.The use of human samples was approved by the Fred Hutchinson Cancer Research Center Institutional Review Board. Fifty study subjects were randomly selected from the 8,506 women assigned to active E+P in the WHI clinical trial, which also included 8,102 women assigned to placebo. All women were postmenopausal, with a uterus, and in the age range from 50 to 79 years, at recruitment during 1993 through 1998. The selected women were required to have been adherent to study medication (80% or more of pills taken) over the first year after randomization, and without a major clinical event  over the intervention and follow-up period (through March 2005). A second nonoverlapping subset of E+P women was selected, by using the same criteria, for replication studies with ELISA. As previously reported [These methods were previously described  in detaiAfter immunodepletion of the six most abundant proteins , pools were concentrated, and intact proteins having cysteine residues were isotopically labeled with acrylamide . The baseline and 1-year pools were than mixed together for further analysis.The combined sample was diluted, and each sample was separated into 12 subsamples by using anion exchange chromatography, and each subsample was further separated into 60 fractions by using reversed-phase chromatography, giving a total of 720 fractions for each original mixed sample. Aliquots of 200 μl from each fraction, corresponding to about 200 μg of protein, were separated for mass spectrometry 'shotgun' analysis.Lyophilized aliquots from the 720 individual fractions were subjected to in-solution trypsin digestion, and individual digested fractions, four to 60 from each reversed-phase run, were combined into 11 pools, giving a total of 132 (12 × 11) fractions for analysis from each original mixed baseline and 1-year pool. Tryptic peptides were analyzed with an LTQ-FT mass spectrometer. Spectra were acquired in a data-dependent mode in a mass/charge range of 400 to 1,800, and the five most abundant +2 or +3 ions were selected from each spectrum for tandem mass spectrometry (MS/MS) analysis.The acquired LC-MS/MS data were automatically processed by the Computational Proteomics Analysis System . DatabasThe relative quantitation of 1-year to baseline concentration for cysteine-containing peptides identified by MS/MS was extracted by using a script designated Q3 ProteinRatioParser , which ct statistic [Protein log-(concentration) ratios were analyzed by first normalizing further, so that the median of the log-ratios is zero for all the proteins identified from a mixed baseline and 1-year sample. Concentration changes after E+P use were identified by testing the hypothesis that the mean of the log-ratios across the (up to 5) mixed samples is zero, by using a weighted moderated tatistic  implementatistic : the logtatistic  was usedt statistic was used to test for a difference in means between the log-ratios for E+P and those for E-alone, with common log-ratio variance for the two preparations.The same method was used to identify proteins for which the 1-year to baseline change in concentration differed between E+P and E-alone. Specifically, a moderated 2 procedure  to identify sets of proteins, defined by biologic pathways, that change concentration with E+P, or that change differentially in the E+P and E-alone project components. This testing procedure takes advantage of the correlation structure among the log-ratios for proteins in a given set. Protein sets were defined by using the KEGG database [We developed a regularized Hotelling Tdatabase ,28.To accommodate multiple hypotheses testing issues, the significance for individual proteins or for biologic pathways, is based on a 5% FDR criterion.P values were obtained by applying t tests to log-transformed 1-year-to-baseline concentration ratios. Log-ratios from ELISA and IPAS were compared to assess discovery platform signals.ELISAs are commercially available for some of the proteins for which evidence emerged of change after E+P use, or of differential change between E+P and E-alone. ELISA tests were applied according to manufacturer's protocols for individual baseline and 1-year serum samples from an additional randomly selected nonoverlapping 50 E+P and 50 E-alone women, for independent validation of leads from the proteomic discovery work. versus 1,054 for E-alone, including 698 that were quantified in both E+P and E-alone analyses.The average age at enrollment for the selected 50 E+P women is 63.2, similar to that for the trial cohort as a whole. Other study-subject characteristics were generally similar also to those for the entire trial cohort . SubjectP < 0.05) of change from baseline to 1-year with E+P or E-alone, or with both. For E+P, 371 proteins were quantified under these quality standards, of which 132 (35.6%) had P < 0.05 as compared with 18.6 expected by chance, and 98 (26.4%) had FDRs <0.05 compared with 94 for E-alone. Of these, 84 had FDR <0.05 for both preparations. Table S1 in Additional file 1 shows estimated 1-year-to-baseline concentration log ratios for all 378 proteins ranked according to the minimum of P values for change with E+P or change with E-alone. Significance levels  are also given for a test of equality of the E+P and E-alone ratios.Protein concentration ratios were further filtered and curated by using stringent standards (see Methods) for protein identification, including a requirement that a protein is quantified in at least two of the 10 IPAS experiments leading to a focus on 378 proteins (IPIs), all but 10 of which were quantified for both E+P and E-alone. A remarkable 169 (44.7%) of these showed evidence  and insulin-like growth factor 1 (IGF1) were both decreased with E-alone and E+P. Insulin-like growth factor-binding protein 1 (IGFBP1) level was increased with both E-alone and E+P. Strong evidence of the E+P effect exists on each of blood coagulation and inflammation, metabolism, osteogenesis, complement and immune response, and cell adhesion. Moreover, the changes (base 2 logarithm of 1-year-to-baseline concentration ratio in Table Table P < 0.05) from each other. For this analysis, we tested the 368 proteins meeting our identification and quantification criteria that were common to both E+P and E-alone. Twenty-six proteins were identified with nominal P values <0.05 for differential change between E+P and E-alone. The list includes proteins involved in insulin growth factor binding and inflammation. Insulin growth factor-binding proteins (IGFBPs) may be affected differently by E+P compared with E-alone. Specifically, three proteins show nominally statistically significant differences with E+P compared with E-alone. IGFBP1 is increased  with E-alone, but the increase is mitigated with E+P . IGFBP4 is increased with E-alone , but not with E+P . NOV, also known as IGFBP9, is decreased with E-alone  and decreased to a greater extent with E+P .Table versus 1-year difference for E+P and E-Alone at FDR < 0.05 by using a regularized Hotelling T2 test. We also tested the equality of log-concentration ratios between the two regimens for proteins in these pathways by using the same test statistic. Two pathways had FDR < 0.05 (Table MMP2) and phospholipase A2 (PLA2G1B)), and a pathway associated with bladder cancer was represented by three proteins , and vascular endothelial growth factor C (VEGFC)). Both pathways had nominal P values of 0.002, with corresponding FDRs of 0.041. MMP2, a collagenase with the ability to break down extracellular matrix proteins, was common to both pathways, and substantially explains the difference between the two regimens for these pathways.In addition to the protein classifications presented earlier, the 368 proteins quantified for both E+P and E-alone were subjected to protein set analysis. In total, 41 KEGG human disease pathways were represented by at least two proteins in this group. Each protein has been quantified in at least two IPAS experiments in both E+P and E-alone. Table P < 0.05 for differential change between E+P and E-alone based on IPAS mass spectrometry findings each had FDR > 0.3, so that many of these may be attributable to chance. We sought to determine whether concordant changes in these proteins can be demonstrated in an independent set of subjects and with independent methods. Figure CP) and ICAM1 may be due to multiple comparison effects in the discovery component or other factors, notably distinct epitope targets by ELISA assays compared with quantified peptides by mass spectrometry.The 26 proteins with nominal versus E-alone changes, because age-related changes would apply equally for the two regimens.These analyses show that 1 year of use of E+P has a profound effect on the serum proteome, with more than a fourth (26.4%) of quantified proteins having FDR < 0.05 for change. Eight proteins with altered levels were further tested in an independent set of samples. ELISA assays of six of the eight proteins showed changes concordant with the mass spectrometry data. The correlation of initial concentration ratios by mass spectrometry with ELISA ratios from an independent set of samples supports the reliability of the protein changes observed. Our previous report on E-alone  providedP values tended to become less significant because of reduction in 'degrees of freedom' for the moderated t tests. Interestingly, after this adjustment, the FDR for NOTCH2 decreased to 0.02. None of the other FDRs in this sensitivity analysis were < 0.05. Aberrant NOTCH signaling has been implicated in tumorigenesis and has been reported to play an oncogenic role in breast cancer [NOTCH2 serum levels with E+P, but not E-alone, could be related to alteration of signaling and increased risk of breast cancer with E+P but not with E-alone. A differential change between E+P and E-alone in IGFBP1 was supported by ELISA data in an independent set of 50 subjects for each regimen  with E alone compared with E+P, whereas the decrease in NOV was relatively greater with E+P. ELISA testing produced trends in these same directions for all three proteins, but only that for IGFBP1 approached statistical significance in the independent set. Collectively, these analyses suggest that progestin may attenuate some of the estrogen-induced increases in IGF-binding proteins.First, consider proteins involved in the insulin growth factor-signaling pathway. The overall pattern Table  is a greIGF-1 (which is primarily of hepatic origin), in agreement with our study findings for IGF-1 levels [IGF1 have been associated with colon cancer risk [IGF-1 is a strong mitogen, and varying levels of free IGF-1 between E+P and E-alone treatment could help explain the increased risk of breast cancer with E+P.It has been previously suggested that medroxyprogesterone acetate has only a weak degree of opposition to the estrogen-induced decrease of total 1 levels . However1 levels ,33, and cer risk . Changesversus E-alone effects on the serum proteome are presented in Table 2-glycoprotein 1 zinc (AZGP1) gene is regulated predominantly androgens and progestins [AZGP1 protein levels with E+P and a decrease with E-alone. AZGP1 has been identified as a potential prognostic marker for early-stage breast cancer and a useful immunohistochemical marker of apocrine cell differentiation in human breast tissue [AZGP1 in E+P compared with E-alone may be associated with increased risk of breast cancer in the former group. Circulating levels of extracellular matrix proteins (collagen α-1 chain (COL1A1), lumican (LUM), and extracellular matrix protein 1 (ECM1) may also be differentially affected by E+P compared with E-alone, whereas MMP2, a metalloproteinase that breaks down COL1A1, may be increased with E+P compared with E. The extracellular matrix plays a variety of physiological roles, many of which are related to cancer, including tumor invasion. Changes in the extracellular matrix with E+P compared with E-alone could also help explain the differences in cancer risks associated with these treatments.In addition to proteins related to the IGF pathway, several other proteins of biologic interest in the context of E+P ogestins ,36. Our t tissue . IncreasMMP2) are thought to participate in atherogenic inflammation [PGLYRP1 participates in recognition of bacteria by neutrophils, but is independently associated with coronary artery calcification and abdominal aortic plaque [PGLYRP1 concentration among women taking E+P versus E-alone suggests a possible mechanistic link.Several proteins listed in Table ammation . The rolc plaque . The difLUM), contributes to variation in proteoglycan composition of arterial intima by location within the human vasculature. Enhanced deposition of lumican has been observed in the intima of the atherosclerosis-prone internal carotid artery compared with the internal thoracic artery, a relatively atherosclerosis-resistant vessel [Other extracellular matrix proteins Table  may alsot vessel . The relF5) binds to multimerin 1 (MMFN1), with high affinity for storage in human platelet granules, and may modulate thrombosis [Our proteomic comparisons Table  may alsorombosis .Some important considerations exist in assessing the effects of estrogens and progestins broadly on the serum proteome. The preparations considered here are conjugated equine estrogen and medroxyprogesterone acetate. Further studies would be needed to determine whether the changes reported here also arise for the other estrogen  and progesterone (norethisterone acetate or levonorgestrel) treatments. Related to this, these substances are taken orally, and the first-pass hepatic metabolism of oral estrogens is known to stimulate a wide variety of proteins, synthesized in the liver. Of the 378 proteins reported here, 73 are included in the liver-specific gene set listed in Hsiao and colleagues   of potential relevance to the less-favorable E+P effects, compared with those for E-alone, on breast cancer, and CHD. Similarly the addition of medroxyprogesterone acetate may also augment the effects of conjugated estrogens on coagulation factors , of potential relevance to a relatively greater elevation in venous thromboembolism with E+P. These and other leads from our proteomic study will benefit from further testing in women who experienced major clinical outcomes and in matched controls from the WHI hormone therapy trials, to evaluate more directly the potential of these protein-concentration changes to contribute to a biologic explanation for observed trial-outcome patterns.In summary, E+P, like E-alone, has a profound effect on the serum proteome and affects multiple pathways that are relevant to observed clinical effects on cancer, cardiovascular disease, and fractures, among others. The addition of 2.5 mg/d medroxyprogesterone acetate to 0.625 mg/d conjugated equine estrogen may have an impact on the IGF pathway proteins and may affect circulating levels of extracellular matrix proteins  or estrogen (E-Alone) exposure for all 378 quantified proteins.Click here for file


Agaricus have worldwide distribution and include the economically important species A. bisporus. Some Agaricus species are inedible, including A. placomyces and A. pseudopratensis, which are similar in appearance to certain edible species, yet are known to possess unpleasant odours and induce gastrointestinal problems if consumed. We have studied the chemical composition of these mushrooms using GC-MS.Mushrooms in the genus A. placomyces and A. pseudopratensis, respectively, including fatty acids and their esters, amino acids, and sugar alcohols. The most abundant constituent in the volatiles and butanol were phenol and urea respectively. We also identified the presence of ergosterol and two Δ7-sterols. In addition, 5α,8α-Epidioxi-24(ξ)-methylcholesta-6,22-diene-3β-ol was isolated for the first time from both mushrooms. Our study is therefore the first report on the chemical composition of these two species.Our GC-MS studies on the volatile fractions and butanol extracts resulted in the identification of 44 and 34 compounds for Agaricus genus, and provide some explanation for the reported mild toxicity of A. placomyces and A. pseudopratensis, a phenonomenon that can be explained by a high phenol content, similar to that found in other Xanthodermatei species.The results obtained contribute to the knowledge of the chemical composition of mushrooms belonging to the  Agaricus have a worldwide distribution, with up to 90 species recorded in Europe. The genus includes the most economically important and commercially cultivated mushroom in the world, A. bisporus (button mushroom) as well as many other edible species [Agaricus species are inedible, including A. placomyces and A. pseudopratensis Bohus, which are similar in appearance to certain edible species, yet are known to possess unpleasant odours and result in gastrointestinal problems if consumed [Mushrooms in the genus  species . Some Agconsumed ,3. To thA. placomyces and A. pseudopratensis. The results can help characterise the species investigated, indicate the presence of some biologically active compounds and shed light upon their reported mild toxicity.In this article, we report the results of the GC-MS analyses of volatile and polar compounds, in addition to the sterol fraction obtained from the fruiting bodies of A. placomyces and over 80% in A. pseudopratensis. Besides phenol, the main groups of compounds in the volatile fractions were fatty acids and their esters. Because the extraction was performed using ethanol, it is possible that the ethyl esters are artefacts. Our findings offer the first analytical proof of the presence of phenol in these two species.The volatile fractions were obtained from the fresh mushrooms' fruiting bodies and analysed as described in the experimental section. The results obtained are outlined in Table Agaricus section, Xanthodermatei, which is characterised by this typical unpleasant odour. It has been suggested that the production of phenol originates from an evolutionary ancestral biochemical shift, also demonstrated by the Agaricus species of the section Xanthodermatei in defence mechanisms [et al. [Xanthodermatei demonstrate higher phenol contents [A. pseudopratensis is a more recently differentiated species than A. placomyces. However, this is only a preliminary hypothesis that will require further more detailed study.The phenol levels detected are not hugely surprising, given the numerous reports of the mushrooms' phenol-like odour, in addition to their belonging to the chanisms . Accordi [et al.  phenoliccontents . For thiA. pseudopratensis. This finding supports the suggestion of the defensive role of phenolics in section Xanthodermatei [A. pseudopratensis.The polar fractions obtained from the fungal species investigated were analysed by GC-MS after silylation. The derivatisation was necessary in order to increase the volatility of the polar fraction, thereby enabling its analysis by gas chromatography. The results obtained are outlined in Table dermatei . AnotherA. placomyces and a mixture of glucitol and mannitol in A. pseudopratensis. The most abundant constituent of the polar extracts was urea. This finding once again confirms that higher fungi of the family Agaricaceae accumulate substantial amounts of urea in their fruiting bodies [Sugar alcohols are amongst the major soluble carbohydrates found in fungi . In the g bodies . It has g bodies .Agaricus species. As expected the predominant sterol in A. placomyces, was ergosterol, although two others were detected, each possessing a Δ7 double bond. In A. pseudopratensis only the latter two sterols were present in substantial quantities, with ergosterol detected in trace amounts.We also investigated the sterol composition of the two 1H- and 13C-NMR) with those reported in the literature [5,7 sterol. It is noteworthy that this type of compound has recently been shown to express inhibitory behaviour against the HTLV-1 virus, in addition to demonstrating cytotoxic activity against human breast cancer cell line (MCF7WT) [Using column and preparative thin-layer chromatography, a crystalline compound was isolated from the extracts of both species and identified as 5α,8α-epidioxi-24(ξ)-methylcholesta-6,22-diene-3β-ol through comparison of its spectral data  .Agaricus, and help provide further explanation for their reported mild toxicity, which we attribute to their high phenol content similar to that of other Xanthodermatei species [The results obtained add to the knowledge on the chemical composition of mushrooms belonging to the genus  species .The macromycetes (age 3 – 5 days), were collected near Plovdiv, Bulgaria, in October 2005.A. pseudopratensis – 105 g; A. placomyces – 245 g) were cut into small pieces and consecutively extracted with ethanol, ethanol-chloroform (1:1) and chloroform. The extracts were combined, water was added and the chloroform layers were removed and evaporated in vacuo to yield 1.88 g of dry residue for A. placomyces and 0.6 g for A. pseudopratensis.The fresh fruiting bodies of macromycetes  was subjected to a four-hour distillation-extraction on Lickens-Nickerson apparatus . The vol-1, with a 10 min hold at 315°C.A portion of the n-butanol extract (5 mg) was dissolved in 50 μL of dry pyridine, before the addition of 75 μL of bis-(trimethylsilyl)-trifluoroacetamide (BSTFA). The mixture was heated at 80°C for 30 min and analyzed by GC/MS. The silylated extract was investigated by GC/MS using the same instrument described above, with a capillary column HP-5 . A helium carrier gas was used. The temperature programmed at 100 – 315°C at a rate of 5°C minIdentification was carried out by searching commercial library databases. Some components remained unidentified, however, owing to both a lack of authentic samples and library spectra of the corresponding compounds.n-hexane – acetone gradient (30:1 – 5:1) to produce several fractions. The third set of fractions, after further purification by preparative TLC , yielded sterol mixtures: 23 mg from A. placomyes and 7.9 mg from A. pseudopratensis, which were analysed by GC-MS. A gas chromatograph (Hewlett Packard 5890) linked to a mass spectrometer (Hewlett Packard 5972) with a capillary column SPB-50  was employed. A helium carrier gas was used with a temperature programme set at 270°C – 290°C at a rate of 4°C min-1 with a 20 min hold. The ion source was set at 250°C with the ionisation voltage at 70 eV.The chloroform extracts was subjected to column chromatography on silica gel with an n-hexane – methyl ethyl ketone 10:1) obtained from both species 5α,8α-epidioxi-24(ξ)-methylcholesta-6,22-diene-3β-ol was isolated (10.4 mg from A. pseudopratensis and 81 mg from A. placomyces) and characterised through comparison of its EIMS, 1H- and 13C-NMR spectra with literature data [From the fourth set of preparative TLC fractions (silica gel, AP performed the extractions, obtained the volatiles and isolated sterol mixtures, as well as participated in the data analysis and interpretation. KA participated in the data analysis and interpretation. EK performed the isolation and structural identification of the epidioxysterols. DA performed the GC-MS analyses. ML and MG participated in the collection and identification of mushroom material. SP conceived the study and helped draft the manuscript. VB participated in the design and coordination of the study, worked on the data analysis and interpretation and helped draft the manuscript. All authors read and approved the final manuscript.


At present, renal grafts are the most common solid organ transplants world-wide. Given the importance of renal transplantation and the limitation of available donor kidneys, detailed analysis of factors that affect transplant survival are important. Despite the introduction of new and effective immunosuppressive drugs, acute cellular graft rejection (AR) is still a major risk for graft survival. Nowadays, AR can only be definitively by renal biopsy. However, biopsies carry a risk of renal transplant injury and loss. Most important, they can not be performed in patients taking anticoagulant drugs.18F-fluorodeoxyglucose (FDG). 3 h after i.v. injection of 30 MBq FDG into adult uni-nephrectomized, allogeneically transplanted rats, tissue radioactivity of renal parenchyma was assessed in vivo by a small animal PET-scanner (post operative day (POD) 1,2,4, and 7) and post mortem dissection. The mean radioactivity (cps/mm3 tissue) as well as the percent injected dose (%ID) was compared between graft and native reference kidney. Results were confirmed by histological and autoradiographic analysis. Healthy rats, rats with acute CSA nephrotoxicity, with acute tubular necrosis, and syngeneically transplanted rats served as controls. FDG-uptake was significantly elevated only in allogeneic grafts from POD 1 on when compared to the native kidney . Renal FDG-uptake in vivo correlated with the results obtained by micro-autoradiography and the degree of inflammatory infiltrates observed in histology.We present a non-invasive, entirely image-based method to assess AR in an allogeneic rat renal transplantation model using small animal positron emission tomography (PET) and We propose that graft FDG-PET imaging is a new option to non-invasively, specifically, early detect, and follow-up acute renal rejection. This method is potentially useful to improve post-transplant rejection monitoring. The number of patients necessitating treatment for end-stage renal disease, and therefore, the number of patients with renal transplants still increases 18F-Fluor-Desoxy-Glucose (FDG) which can be measured by positron emission tomography (PET) AR of renal grafts is characterized by recruitment of activated leukocytes into the transplant in vivo were compared to FDG-activity of renal parenchyma in micro-autoradiography and in transplants to histological changes of acute renal rejection  ex vivo.The aim of this study was to establish and validate an entirely image-based method for the early detection and follow-up of renal allograft rejection in rats in order to translate this approach to human patients in the future. It is based on high-resolution whole body small animal PET following i.v. injection of FDG. FDG-uptake of renal parenchyma was assessed in uni-nephrectomized, allogeneically kidney-transplanted animals (aTX) with, and additionally in native controls (CTR) and syngeneically transplanted (sTX) animals without rejection or impairment of renal function. Because acute cyclosporine A nephrotoxicity (CSA) is a reversible process in which renal function can recover if the drug administration is stopped at the early stage of the disease, differential diagnosis of this disease with other renal dysfunctions that occur after kidney transplantation is of interest. In particular, acute tubular necrosis (ATN) caused by ischemia/reperfusion injury during the transplantation procedure shows a similar clinical course. Therefore, we included two additional groups into the study. One group with acute CSA nephrotoxicity and another with renal ischemia/ reperfusion damage developing acute tubular necrosis (ATN). PET results 2 = 0.69, In allografts undergoing AR, we detected a clearly elevated FDG-uptake starting on postoperative day 1 (POD) , when co18F-fluoride clearance in a subgroup of animals to exclude decreased renal function as a potential cause for increased FDG-uptake into the renal parenchyma. FDG-uptake (%ID) did not correlate to 18F-fluoride-clearance  of the reference kidney was compared to the radioactivity of the graft. POD 4 was chosen for autoradiography, because FDG-uptake in allografts peaked at this time point in PET, nonetheless the integrity of the graft was still intact as confirmed by histology , medulla (15.8±0.5%) and cortex (76.9±0.9%) as confirmed by micro-dissection (n = 14). In PET, FDG-uptake of renal parenchyma was measured with exclusion of renal pelvis. Therefore, theoretically 17.1±0.6% of the signal measured by PET can be attributed to the medulla and 82.9±0.6% to the cortex. Hence, the key question was still, if the FDG-uptake of medulla and cortex equals. Discriminating medulla and cortex for certain can only be performed, if visually confirmed. Therefore, we have chosen to investigate FDG-uptake of medulla and cortex by autoradiography. In native kidneys and syngeneic grafts, the renal medulla possesses an FDG-uptake, which is 1.88±0.18 (CTR) or 1.98±0.08 (sTX) times higher than the matching renal cortex, respectively . TherefoThe FDG-ratio of medulla and cortex (MC-ratio) changes in kidneys undergoing AR. In contrast to graft medulla, the cortex of aTX revealed a distinct higher FDG-uptake than the renal cortex of CTR and sTX. Therefore, the MC-ratio of aTX dropped to 1.39±0.08. As confirmed by HE-staining (data not shown), a lower ratio was linked to a more intense infiltrate in the cortex than in the medulla. In summary, we found that areas with increased FDG-uptake coincide with areas of inflammatory cell infiltrate.Signs of acute rejection are glomerulitis, tubulitis, endothelialitis, and graft infiltration 2 = 0.96).To verify our hypothesis that the uptake of FDG (%ID) is directly related to the degree of inflammatory infiltration and thereby to the grade of AR, we correlated the leukocytes/FOV of each group  to their according %ID . This coWe evaluated the ability of FDG-PET to discriminate between kidneys from different control models  and individuals with acute allograft rejection by a Receiver Operating Characteristic (ROC) curve. enhanced FDG-uptake (cpm/mm2/MBq and %ID) in transplants undergoing rejection to reference methods, i.e., to the histological quantification of infiltrating leukocytes  ratio of FDG-uptake. This MC-ratio was identical to the MC-ratio of syngeneic transplants, but significantly decreased in rejected allografts . One reaFunctional imaging with FDG is widely used (and therefore available in common) in clinical setups, e.g., diagnosis and staging of malignant disease, in image-guided therapy planning, and detection and grading of inflammation, be it infectious, autoimmune, or of other origin One might worry about the exposure to radiation when applied to humans, but the dosage applied, is rather low and close to other clinical investigation techniques, e.g., lower than the dosage which is applied during an abdominal computer tomography. In addition and in contrast to several contrast media used in computer tomography or magnetic resonance imaging, the tracer is neither toxic to kidney nor carries the risk of nephrogenic systemic fibrosis.A relevant diagnostic problem in case of graft dysfunction might be the discrimination of AR and CAF. CAF also featuring a certain degree of allograft infiltration is the most common reason for graft loss In conclusion, we present and validate a non-invasive, entirely image-based method to assess acute renal rejection using FDG-PET. We suggest that this method may have a potential clinical impact for the diagnosis of AR and may be useful to discriminate AR from ATN and acute CSA-induced nephrotoxicity, in patients with severe contraindications to perform graft biopsy.Male Lewis–Brown-Norway (LBN) and Lewis (LEW) rats  with free access to standard rat chow  and tap water were used. Experiments were approved by a governmental-committee on animal welfare  and were performed in accordance with national animal protection guidelines. Surgeries were performed under anesthesia with ketamine 100 mg/kg body weight intra peritoneal (i.p.) and xylazine 5 mg/kg BW i.p. . Further doses of ketamine were injected as needed. Transplantation was simultaneously performed by two investigators as published before 18F-fluoride and FDG were produced on site using an RDS 111 cyclotron . The FDG-uptake was calculated from a whole body acquisition of 30 min length in a high-resolution small animal PET scanner 3 hours after i.v. injection of 30 MBq FDG in 100 µl 0.9% NaCl solution in a tail vein.Clinical grade Afterwards the catheter was purged with additional 900 µl 0.9% NaCl solution. The animals remained in a restrainer without anaesthesia until start of the scan and were hydrated by i.v. injection of 1000 µl of 0.9% NaCl solution hourly. Acquisition started 3 h after FDG-injection to reduce tracer uptake in the kidneys caused by renal excretion of FDG.During acquisition, rats were anaesthetized with oxygen/isoflurane inhalation  and body temperature was maintained at physiological values by a heating pad. PET scans were performed with the high-resolution multi-wire chamber-based animal PET camera quadHIDAC . The field of view (FOV) of the PET scanner is 28 cm axially and 17 cm in diameter. The spatial resolution of the PET scanner is <1.0 mm and is constant over the whole FOV 18F-fluoride without motion of the position of the rat in the scanner. Another PET acquisition of 15 min length was immediately initiated after 18F-fluoride injection for identification of renal parenchyma  or of 60 min for measurement of fluoride clearance.The FDG-PET acquisition was followed by i.v. injection of 5 MBq 18F-fluoride injection was transferred to the FDG images. The renal pelvis was carefully excluded from the VOI. Mean FDG-uptake of the renal parenchyma was calculated by the ratio of total counts and volume.A renal parenchyma VOI manually traced around the kidneys on reconstructed images 2 min after Renal fluoride clearance and spilt renal function was calculated by the ratio of the renal excreted activity 60 min p.i. and the integral of the blood pool time activity curve in a subgroup of acquisitions (n = 22) as recently described 2) was measured for 3 h. One region was defined as the medulla-region, the other as the cortex-region. A ratio medulla/cortex (MC-ratio) was calculated. Additionally, a more “realistic” mean whole kidney-radioactivity , was calculated as followed: (mean activity of medulla [cpm/mm2/MBq+mean activity of cortex [cpm/mm2/MBq] * 3)/4.To validate the signal obtained by PET animals were euthanized and the kidneys were harvested immediately after FDG-scanning on POD 4. High-resolution micro-autoradiography  was performed on cryosections (10 µm thick) of snap-frozen transplant or normal kidney. Two regions of interest were visually identified and mean activity  were examined by light microscopy in paraffin-embedded tissue with hematoxylin–eosin (HE) staining. Graft infiltration was quantified according to the ti-score . Laboratory data was compared with ANOVA variance analysis for multiple comparisons where appropriate. Data is presented as mean values±SEM . Significance was inferred at the p<0.05 level.